Evaluation of calcium and phosphorus elements in the developing human fetal long bone at different trimesters of pregnancy

Abstract

Recent bone research has emphasized the importance of mineral composition and peak bone mass during the fetal and postnatal periods, impairment in which are related to subsequent bone diseases in later life. However, there is no quantitative data on the variations in the composition across the growth period of fetal bone to gain better insight into the fetal origin of adult bone disorders and old-age bone diseases. This study analyzed the mineral composition of the developing humerus bone in different trimesters of pregnancy. Stillbirth or medically terminated human fetal specimens ranging from approximately 10–36 weeks were obtained after institutional ethical committee clearance. The transverse section of each humerus close to mid-diaphysis was subjected for mineral analysis by scanning electron microscopy and energy dispersive spectroscopy techniques. The results indicated that the calcium (Ca) noticeably varies between 10 and 20 weeks and 24 and 36 weeks. It is also noted that the mean phosphorus level remained constant from 12 to 36 weeks. Further, significant differences were observed in Ca to phosphate ratio at different gestational weeks. The study provides preliminary quantitative insight into fetal cortical bone mineral composition and may serve as a foundation for future investigations examining broader anatomical regions, significance of mineral constituents, and functional correlations.

Introduction

Over the past few decades, there has been a lot of activity in the field of research into the microscopic and mechanical properties of bones [1-3]. The effects of aging [4], illnesses like osteoporosis [5], cancer [6], and other bone diseases, as well as the micro-level organization and mineral composition of bone, have all been better understood through literature [7, 8].

Bone tissues comprise both major components, calcium (Ca), phosphorus (P), magnesium (Mg), and sulphur, and trace components such as iron, zinc, manganese, and cadmium. Most of the elemental research has centred on the primary elements—notably Ca, P, and Mg—due to their vital functions in bone metabolism [1, 2]. Despite the ossification centre appears at the future diaphyseal region of a long bone between 8 to 12 weeks of intrauterine life, the process of mineralization at the centre begins from 3rd month. Though Ca contributing much to attain peak bone mass in early life, P is also another primary element in bone tissue. Extracellular Ca and P ions are the key elements in mineralization process of bone as determined by experiments on vitamin D3 signalling pathway. Additionally, P ions are also shown to have more critical value in mineralization than Ca ions as found in case of osteomalacia by CaSR and Phex gene mutation in Hyp mice. Decreased levels of P ions is shown to induce mineralization defects in the bone tissue, despite the normal Ca levels [9]. Thus, appropriate concentration of Ca and P is key in maintaining mineral composition and bone health. Disturbed Ca-to-P (Ca:P) ratio concentrations are proved to be detrimental to bone health consequences.

The total amounts of Ca and P in a human adult body is about 99% and 80% respectively. Concentration of Ca and P minerals endowed with phosphatase enzyme activity are an integral part of matrix formation by strengthening the network of collagen fibres, which is necessary to support mechanical loading of a bone. The mineralization process necessitates a “boosting” mechanism that amplifies the concentration of Ca and P minerals inside the skeletal milieu. The initial stage of Ca:P crystal nucleation occurs inside matrix vesicles, which emerge from osteogenic cells’ plasma membrane and move into the extracellular skeletal compartment [10]. Excess extracellular concentration of Ca promotes bone formation and inhibits bone resorption though its activity on bone cells, mediated by Ca-sensitive receptor. Thus, Ca supplementation has a positive impact on bone mass [11].

The fetal bone development is viewed as a process of bone mineral growth or increase in bone mass [12]. The knowledge of the mechanisms of mineral accumulation in bones, and the concentration and distribution of elements in the bone tissue has not so far been fully understood [3]. Scanning electron microscope (SEM) and energy dispersive spectroscopy (EDS) are mostly preferred to study pattern of bone growth, elemental composition, distribution and concentration of various minerals within the bone sample with accurate data at micro- and nanoscales. Earlier studies mentioned that the mineral composition of bone tissue can be assessed by non-invasive and non-destructive methods, such as nuclear magnetic resonance, spectroscopic imaging (Raman Imaging and Fourier Transform Infrared), scanning electron microscopy, and destructive gravimetric analysis using bone ash weight [13].

Currently, no study has described the sequential changes in the mineral composition of the developing human fetal humerus bone at different trimesters of pregnancy. Thus, the present preliminary study was designed to evaluate changes in the mineral composition of the developing cortical portion of the humerus in each trimester, close to the mid-diaphyseal region, using SEM and EDS techniques.

Materials and Methods

Specimen collection

Human fetal specimens ranging from approximately 10–36 weeks (two specimens from each trimester) were obtained after medically terminated or spontaneous abortions or stillbirths. The informed written consent from the patients followed the tenets of the Declaration of Helsinki and Institutional Ethical Committee clearances (Ref no: IEC-121/2016, from Institutional Ethical Committee Registration No. ECR/146/Inst/KA/2013) and as per the guidelines of Manipal Academy of Higher Education. Fetal humeri from 10 and 12 weeks (for the first trimester), 20 and 24 weeks (for the second trimester), and 32 and 36 weeks (for the third trimester) were collected from the Department of Obstetrics & Gynaecology, affiliated to Kasturba Medical College, Manipal.

Inclusion and exclusion criteria

Fetal specimens without any clinical, radiological, and morphological evidence of musculoskeletal disorders were included in this study. Disarticulated humerus bones from fetuses were stored in formaldehyde solution in a completely hydrated state. The soft tissues attached to the bone were removed and cleaned using deionized water. Subsequently, small sections of cortical bone at the mid-diaphysis region (≈5 mm) were cut using a scalpel and stored in phosphate-buffered saline at –20°C.

Risk of bias assessment

All accessible fetal humerus specimens that satisfied the eligibility requirements during the study period were included in the “Joanna Briggs Institute’s” critical evaluation checklist for analytical cross-sectional research. Because the sample does not reflect a random distribution of the fetal population, this approach may induce selection bias. Because samples from medically terminated, spontaneously aborted, or stillbirth fetuses may not precisely reflect the nutritional or developmental condition of healthy live births, their use may restrict the findings’ generalizability. Additionally, sample sizes varied across trimesters due to difficulties in acquiring specimens at particular gestational ages, which may have an impact on the statistical power of comparisons.

SEM imaging and EDS analysis

The mid-diaphysis region of the bone specimens was fixed on stubs using adhesive tape, with cortical bone facing upward. The exposed surfaces of the specimens were sputter-coated with gold (SC7620 Sputter Coater; Quorum). Specimen surface features were examined under vacuum using a SEM (EVO-MA18; ZEISS). Analysis was conducted using a specific SEM Control User Interface software.

Each specimen was subjected for mineral composition using Oxford Instruments X-ACT with SEM. Each analysis was performed after selecting an area of 400 µm² on standard SEM photomicrographs acquired at 20 kV, as shown in Fig. 1. Quantitative analysis was performed using the INCA Energy Software Interface of Oxford Instrument, which assesses the absorption, atomic number, and fluorescence of the minerals present in the sample areas. The weight percentage (Wt%) of identified minerals determined during the test was used for further analysis.

Fig. 1.

A scanning electron microscope imaging of a specific location of fetal bone and energy dispersive spectroscopy analysis in the same region indicates the presence of various elements in bone tissue.

Method of analysis of minerals

In the present study, the total number of fetus/humerus specimen n=6, in each specimen tests on separate 7 places were conducted to capture cortical EDS measurements at different locations. This is due to capture local mineral heterogeneity as no individual site within the bone is same with respect to the mineral presence and composition. The seven EDS measurements per specimen were averaged to generate a single specimen-level mean value for Ca and P. The Ca:P ratio was calculated using mean Wt% values of Ca and P.

These specimen-level means were then used for all statistical comparisons across trimesters. As shown in Tables 1 and 2, the mean of seven sites per specimen was calculated first. These specimen-level means were then used for; one-way ANOVA across trimesters and Bonferroni post hoc comparisons (for Ca:P ratio) as analysed in Table 3. The Ca:P ratio represents a derived variable reflecting the proportional balance between Ca and P. While individual elemental Wt% did not show statistically significant trimester-wise variation, modest coordinated shifts between Ca and P may produce detectable differences in their ratio.

Table 1.

Calcium weight and atomic percentages at different sites in the mid-diaphyseal region of the developing human fetal humerus at different weeks of pregnancy

No. of the sites analyzed	Ca
	10 weeks			12 weeks			20 weeks			24 weeks			32 weeks			36 weeks
	Wt%	At%		Wt%	At%		Wt%	At%		Wt%	At%		Wt%	At%		Wt%	At%
1	8.87	3.14		16.22	6.73		14.68	5.91		18.42	7.87		16.58	6.86		20.61	8.98
2	15.07	5.94		64.45	58.14		63.23	57.59		64.74	59.29		63.94	58.23		67.36	61.48
3	66.62	61.34		14.87	6.12		17.78	7.57		20.53	8.85		63.94	58.23		67.36	61.48
4	11.16	4.17		61.44	55.74		63.47	57.61		64.37	58.61		15.23	6.24		20.07	8.72
5	64.77	59.31		61.39	55.73		63.47	57.61		21.38	9.48		64.80	59.23		65.46	59.61
6	67.51	61.57		12.34	4.91		16.57	6.98		21.38	9.48		16.62	6.89		19.19	8.11
7	8.64	3.06		61.93	55.83		63.01	57.32		64.77	59.33		63.31	58.07		64.52	59.22
Mean±SD	34.66±27.47	28.36±28.06		41.80±23.71	34.74±24.97		43.17±23.25	35.79±25.10		39.37±21.89	30.41±24.82		43.48±23.68	36.25±25.62		46.36±22.89	38.22±25.66

Ca, calcium; Wt%, weight percentage; At%, atomic percentage.

Table 2.

Mean weight percentages of each mineral from seven different sites on the cortical portion of the mid-diaphyseal region of human fetal humeri (n=6)

No. of the sites analyzed	P
	10 weeks			12 weeks			20 weeks			24 weeks			32 weeks			36 weeks
	Wt%	At%		Wt%	At%		Wt%	At%		Wt%	At%		Wt%	At%		Wt%	At%
1	4.08	1.87		9.20	4.94		7.88	4.10		9.59	5.30		9.25	4.95		10.17	5.74
2	7.23	3.69		34.27	40.00		32.63	38.46		32.11	38.05		33.73	39.75		31.47	37.16
3	30.77	36.67		8.75	4.67		10.01	5.52		11.01	6.15		33.73	39.75		31.47	37.16
4	5.54	2.68		34.20	40.15		33.71	39.59		32.85	38.71		8.61	4.56		10.55	5.94
5	31.04	36.79		34.17	40.14		33.71	39.59		11.10	6.37		33.82	40.00		32.61	38.42
6	30.16	35.59		7.17	3.70		9.31	5.08		11.10	6.37		9.17	4.92		9.94	5.44
7	3.98	1.82		34.11	39.79		33.48	39.40		32.02	37.95		33.15	39.35		32.11	38.14
Mean±SD	16.11±12.63	17.01±16.75		23.12±12.78	24.77±17.61		22.96±12.05	24.53±17.01		19.96±10.71	19.84±15.93		23.06±12.17	24.75±17.27		22.61±10.74	24.00±15.84

P, phosphorus; Wt%, weight percentage; At%, atomic percentage.

Table 3.

Bonferroni’s multiple comparison test for calcium-phosphate ratio across different gestational ages

Bonferroni’s multiple comparison test (wk)	Mean difference	t	Significant at P<0.05	Summary	95% confidence interval
10 vs. 12	0.3514	9.470	Yes	***	0.2348 to 0.4681
10 vs. 20	0.2757	7.430	Yes	***	0.1590 to 0.3924
10 vs. 24	0.1843	4.966	Yes	***	0.0676 to 0.3010
10 vs. 32	0.2771	7.468	Yes	***	0.1605 to 0.3938
10 vs. 36	0.1100	2.964	No	NS	–0.0067 to 0.2267
12 vs. 20	–0.0757	2.040	No	NS	–0.1924 to 0.0410
12 vs. 24	–0.1671	4.504	Yes	**	–0.2838 to –0.0504
12 vs. 32	–0.0743	2.002	No	NS	–0.1910 to 0.0424
12 vs. 36	–0.2414	6.506	Yes	***	–0.3581 to –0.1248
20 vs. 24	–0.0914	2.464	No	NS	–0.2081 to 0.0252
20 vs. 32	0.0014	0.038	No	NS	–0.1152 to 0.1181
20 vs. 36	–0.1657	4.466	Yes	**	–0.2824 to –0.0490
24 vs. 32	0.0929	2.502	No	NS	–0.0238 to 0.2095

NS, not significant. **Significant; ***highly significant.

Results

Cortical and trabecular bones at mid-diaphysis in different trimesters

Histomorphometric data of the developing fetal bone in the mid-diaphysis region at each trimester of pregnancy (Fig. 2) are presented as follows. (1) First trimester: the periosteum covering the bone is very thin as a collar around the developing bone. The mineralized bone had a thin cortical segment and indistinct trabeculation in the central cancellous area, as seen in 12 weeks. The bone marrow medullary cavity appeared larger than the cortical area. (2) Second trimester: the outer cortical bone is gradually attained thickness with well-developed osteons. The marrow cavity was smaller with immature trabeculation as shown in 20 weeks. The bone marrow medullary cavity proportionally appeared less than the cortical segment. (3) Third trimester: thick and concentric arrangement of bony lamellae was observed close to the periosteum and marrow cavity. The cortical bone has reached its maximum width with interlacing less trabeculation in the marrow cavity, as observed in 32 weeks.

Fig. 2.

SEM images of the cortical bone surface of the humerus. (A) 12 weeks (first trimester), (B) 20 weeks (second trimester), and (C) 32 weeks (third trimester).

Mineral analysis of the bone

Seven cortical (compact) bone sites were identified by SEM in each specimen and subjected to EDS analysis for estimation of mineral contents (Fig. 1). In the EDS analysis, the Wt% and atomic percentage (At%) were used to express the composition of the elements in a sample. Wt% provides information about the relative abundance of elements in terms of their weight contributions. Similarly, the At% represents the proportion of elements expressed as percentage of total number of atoms present in it. Tables 1 and 2 presents the mean Wt% (calculated as the mean weight of seven different sites in each sample) of Ca and P elements found in the cortical bone at mid-diaphysis after EDS analysis.

Mean Wt% of Ca is 34.66 and 41.80 in the early and late 1st trimester respectively (Table 1). Later, the mean Ca remains constant with minimal change due to the remodelling process of bone until the end of the 3rd trimester where it assumes highest (46.36 wt%). The mean Wt% of P ion ranges from 16.11 wt% in the early 1st trimester to 22.61 wt% in the late 3rd trimester with a minimal variation in the rest of the growth phase. Like the Ca, even in P ion the minimal variations are attributed to bone remodelling process across the trimesters (Table 2). The statistical evaluation of Ca and P mineral across the trimesters yielded P-value of 0.971 and 0.881 respectively, which is not significant. Individual variations among each element across the three trimesters were not significant due to larger variations of each element at each site of EDS analysis. Absolute Ca and P Wt% did not demonstrate statistically significant trimester-wise variation. In contrast to individual elemental values, the Ca:P ratio demonstrated statistically significant differences across gestational ages, suggesting proportional shifts in mineral balance. However, these proportional changes reflected in the Ca:P ratio may indicate subtle shifts in mineral stoichiometry during cortical bone maturation.

Ca:P ratio was 2.13 and 1.78 in the first trimester, 1.85 and 1.94 in the second trimester, and 1.85 and 2.02 in the third trimester. One-way ANOVA was performed to verify the statistical significance of mean weight ration of Ca and P across the three trimesters and Bonferroni’s multiple comparison test was performed to lessen the type 1 error (Fig. 3).

Fig. 3.

Graphical representation of the Ca:Po₄ ratio in the fetal cortical bone of different gestational ages. P>0.05 (*In comparison to 10 weeks; ^#in comparison to 12 weeks; ^$in comparison to 20 weeks; ^@in comparison to 32 weeks).

Discussion

Mineralization of the developing bone in fetal life begins through the formation and growth of bioactive crystallites at the site of the future diaphysis of the long bone [14]. These phases are crucial in determining bone quality in later life stages. However, a handful of questions remain related to bone mineral appearance and growth during bone formation. Skeletal dysplasia is associated with defects in the production or functionality of bone mineral matrix. This results in compromised skeletal growth due to excessive bone tissue [15]. There has been an increasing shift toward the emergence and use of biomaterials for repairing and restoring severely injured bone, and biomaterials have been initiated to promote new bone formation when implanted in the injured bone [16].

The intricate structure of bone arises from the presence of dissimilar organic and inorganic components. The inorganic components of bone are mainly hydroxyapatites, which form the mineral phase of bone. Organic components, primarily consisting of cells, collagen, and non-collagenous proteins, contribute to bone structure, flexibility, and tensile strength [17, 18]. The intake/supplementation of minerals strongly influences bone composition and development. Traditional minerals like Ca and P play various roles in the formation and structure of hydroxyapatite crystals. Other nutrients play secondary roles as co-factors or regulators of cellular activity during the development of bones [19]. Earlier studies have emphasized the importance of mineral composition and peak bone mass during the fetal and postnatal period, which are related to subsequent skeletal health issues such as osteopenia, osteoporosis, and fracture in later stages of life [20].

In a prospective study, maternal Ca supplementation at mid-gestation was significantly associated with the length of long bones of fetus. Although the exact mechanism is not clearly defined, Ca positively affects the development of fetal bones through excess collagen synthesis, increased cell lineage rate, and differentiation at the growth plate region [21]. In his review, Zwanziger mentioned that the complicated matrix of bone has Ca and P as primary measures of bone mineral content [22]. Ca influences intracellular and extracellular processes essential for bone development, growth, and maintenance. Inadequate Ca supply during bone development in fetal life may hinder bone maturity, predisposing individuals to an increased risk of osteoporotic fractures later in life [23]. Compared to the Wt% of Ca in the early 1st trimester, which is very low and picked up towards the end of the 1st trimester. Thereafter the Wt% of Ca maintained same till the early 3rd trimester. Probably it could be due to the remodelling phenomenon that happened throughout the gestation period. Ca accretion is maximum between the early and late periods of 3rd trimester.

The element P is another critical constituent of the inorganic matrix of bone that plays a crucial role in the formation of hydroxyapatite crystals. Phosphate ions combine with Ca ions to form hydroxyapatite crystals, which in turn provide strength and rigidity to bone. An adequate amount of phosphate is crucial for chondrocyte apoptosis in the growth plate. In the absence of a sufficient quantity of this mineral, chondrocyte will not undergo apoptosis, and the expected physiological processes, including blood vessel invasion and the development of new bone, will be altered, resulting in rickets and delayed growth [24]. In the current study, concentration of P followed a similar pattern as Ca, as shown in Tables 1 and 2. Because P is primarily entangled with Ca in the form of hydroxyapatite crystals, the remaining mineral appears as formless Ca-phosphate. The Ca:P ratio governs the absorption, retention, and/or balance of Ca and P in the body through regulatory processes. From fetus to adult, ranges from 0.58 to 2.34 (mg:mg). Any Ca:P ratio imbalance impairs the process of bone remodelling and makes the fetus more vulnerable to osteoporosis in adulthood and stunted growth in childhood [25]. Present study on the developing long bones showed Ca:P ratio varied from 1.7 to 2.15 (Fig. 3). However, the Ca:P ratio in adult humans ranged from 1.3 to 2.2 as recorded in previous studies [26]. Also, any conditions that affect the concentrations of either P or increased parathyroid hormone (PTH) will potentially contribute to an abnormally low Ca:P ratio and net loss in bone mass [27] as depicted in below (Table 4).

Table 4.

Effect of dietary calcium and phosphorus on the bone mass

Dietary intake	Intestinal absorption	PTH secretion	Effect on bone mass
Low Ca, low P	Decreased Ca absorption	Increased	Decreases by increasing resorption
Low Ca, high P	Decreased Ca absorption & increased P absorption	Increased	Decreases by increasing resorption
High Ca, low P	Increased Ca absorption	Increased	Mineralization defects due to poor apatite crystal formation

Ca, calcium; P, phosphorus; PTH, parathyroid hormone.

In continuation the above observations, the mechanical loading and mineral homeostasis has been sensed well by the osteocytes through its vast canalicular network that spreads over the entire bone surface area. Osteocytes produce both promotors and inhibitors of bone mineralisation. The promotor like dentine matrix protein 1 by the osteocytes and P mineral promotes bone mineralization process through osteoblasts [24, 28]. Thus, phosphates exhibit new bone formation by their bioactive properties (like osteoinductive and osteoconductive) on the osteoblast’s adhesion and proliferation. This was proved with osteomalacia induced on CasR and HyP mice with Phex gene mutation. This could be since the serum Ca level is more tightly regulated than that of P as the former is critical in numerous physiological activities [29].

Ca phosphates, composed of Ca and P ions have been used as implantable biomaterial in bone regeneration applications. Studies proved that Ca phosphates can be used as surface coatings, cements, or scaffolds to enhance the bioactivity of bone transplants and fracture healing in orthopedics and faciomaxillary surgeries [30]. Also, the supplementation of Ca:P mineral is effective in early prevention of adult osteoporosis, fragility fractures, and osteopenia [31]. Minor bone defects exhibit limited self-regeneration, while major bone defects necessitate autologous or allografts. An artificial bone graft with all the characteristics of natural bone tissue is necessary for the effective treatment of bone defects. Thus, the creation of safe and efficient materials or composites—like artificial bone substitutes—is necessary for bone tissue regeneration, and this has sparked a lot of interest in transforming the clinical management of bone disorders and abnormalities [32, 33].

Limitations and future scope

The specimens were obtained from medically terminated pregnancies, spontaneous abortions, and stillbirths, the findings may not fully represent mineral composition patterns of healthy live-born fetuses. Although no skeletal abnormalities were identified in the included specimens, unrecognized systemic or developmental factors cannot be entirely excluded. Therefore, the results should be interpreted as descriptive data from available fetal specimens rather than definitive reference values for normal fetal development.

The present findings are limited to elemental characterization of fetal mid-diaphyseal cortical bone. Larger-scale investigations integrating mechanical testing, additional mineral constituents, and longitudinal developmental analyses would be necessary before drawing translational conclusions related to adult bone pathology. The understanding of early-life mineral composition will contribute to broader developmental models of skeletal health.

Conclusions

The observed trimester-wise patterns represent compositional findings within the analyzed specimens. This exploratory study characterizes the Ca and P composition of the mid-diaphyseal cortical region of the developing human fetal humerus across trimesters. Individual Ca and P levels did not demonstrate statistically significant trimester-wise variation, proportional changes reflected in the Ca:P ratio suggest potential shifts in mineral stoichiometry during fetal bone maturation. The study provides preliminary quantitative insight into fetal cortical bone mineral composition and may serve as a foundation for future investigations examining broader anatomical regions, additional mineral constituents, and functional correlations.